Epithelial cells of the gastrointestinal tract serve as a selective barrier to the diffusion of macromolecules between tissue compartments. However, the transepithelial transport of a subset of molecules is required for some intestinal functions, particularly those related to mucosal immunity. The best characterized example of transcytosis is that of the polymeric immunoglobulins IgA and IgM which are the predominant antibodies in mucosal secretions. These molecules, which form the first line of defense against mucosal pathogens, undergo receptor-mediated transport across the epithelium, a process that requires the passage of receptor-ligand complexes through several subcellular compartments, using a number of vesicular intermediates. It is the overall objective of the proposed research to understand the molecular mechanisms by which proteins are internalized and packaged for subsequent transport across epithelial cells. A common feature of vesicular transport processes is the involvement of small GTPases which serve as molecular switches to control the assembly, targeting and fusion of carrier vesicles. Two subclasses of this family have been directly implicated in specific transport steps: ADP- ribosylation factors (ARFs) regulate vesicle budding, whereas rabs regulate vesicle docking and fusion. This proposal will examine the role of two of these proteins, rab25 and ARF6 in transepithelial transport. Unlike other rabs that are ubiquitously expressed, rab25 is expressed exclusively in epithelia, suggesting that it regulates a transport process that is unique to epithelia. Consistent with this hypothesis, we have localized rab25 to an apical endosomal compartment, through which IgA passes en route to the apical surface. Using MDCK cells as model epithelium, we will delineate the role of rab25 in the transcytotic pathway, by expression of dominant inhibitory mutants using adenoviral expression vectors. (Aim 1). Specific effects of mutant protein expression on immunoglobulin transport will be correlated with morphological consequences by both confocal and electron microscopy. ARF6 has been shown to regulate endocytic processes in non-polarized cells. We have found that, while ARF6 is expressed at both apical and basolateral poles of the cell, it appears to selectively modulate apical endocytosis and recycling. We will determine the specific transport steps regulated by ARF6 and correlate these with effects of the protein on the actin cytoskeleton (Aim 2). GTPases require the participation of accessory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) to complete the GDP-GTP cycle. We have identified a GEF that is selective for members of the ARF family, and in vivo appears to be specific for ARF6. This protein is homologous to a yeast protein, sec7, and contains a pleckstrin homology (PH) domain, which regulates the catalytic activity of the protein in response to the binding of inositol phospholipids. In keeping with our observation that ARF6 selectively regulates apical endocytic processes, we have found that this GEF is localized to the apical pole of several types of epithelia in vivo. We will determine the functional role of this protein in apical endocytosis, and will biochemically biochemical characterize its functional domains (aim 3). These studies will provide insight into the mechanisms by which macromolecules are transported across epithelia, a process that is particularly important in the mucosal immunity.